The long-term objectives of this project are to define molecular events underlying cell-associated (CA) HIV-1 transmission across human cervical and vaginal epithelia, and to develop an in vitro model for testing the effects of vaginal microbicide formulations on CA HIV-1 transmission. The aims of the first (R21) phase of this project are to define adhesion molecule receptor/ligand combinations that play a role in the attachment of HIV-1 infected cells to human cervicovaginal epithelial cells, and chemokine/ chemokine receptor (CR) combinations that promote penetration of infected cells into the vaginal epithelium. We will use multichannel cytofluorometric analysis to characterize adhesion molecule and chemokine receptor (CR) expression on CD4+ lymphocytes and macrophages isolated from semen of HIV-infected men, and immunohistochemistry and Bio-Plex/ELISA assays to characterize the expression of complementary adhesion molecules and chemokines by cells in human vaginal and cervical tissues. We will perform parallel studies on CD4+ lymphocyte and macrophage cell lines (HIV+ seminal cell surrogates) and on the MatTek EpiVag organotypic model (human vaginal epithelium surrogate) to validate their appropriate expression of these molecules as the first step towards establishing an authentic in vitro model of CA HIV-1 sexual transmission. In Phase II (R33) we will use the MatTek EpiVag model to further define mechanisms of CA HIV vaginal transmission, and to develop and validate a quantitative CA HIV transmission assay. We will use this assay to test the efficacy of vaginal microbicide formulations and to screen for other factors that inhibit CA HIV transmission. This model system could accelerate the development of vaginal microbicides to prevent the sexual transmission of HIV-1, and lead to the identification of novel CA HIV blocking factors that could enhance the efficacy of vaginal microbicides.